RNA modifications are ubiquitous in biology. In ribosome, they regulate its structure and functions, modulate antibiotic drug resistance, and have been implicated in human diseases. Recent successes in identification and structural localization of rRNA modifications facilitate mechanistic studies of RNA modifications during the ribosome biogenesis. While most of the prior efforts have been focused on characterization of structure and specificity of modification enzymes in vitro, how individual rRNA modifications are integrated in the complex multistep biogenesis process remains unclear. The proposed study will establish the utility of a modern mass spectrometry methodology and stable isotope labeling for quantitative analysis of rRNA modifications, and apply this technique to decipher the timing and regulation of rRNA modifications during ribosome assembly in E. coli. To place RNA modifications into the existing framework of ribosome assembly we will use bacteria genetics to introduce perturbations at different points on the assembly pathway via systematic depletions of individual ribosomal proteins, biogenesis factors and modification enzymes. Then, mass spectrometry will be applied to characterize protein composition and rRNA modifications in the accumulated assembly intermediates to reveal patterns of relationships between individual rRNA modifications and other assembly steps. By providing new mechanistic insights into the entire rRNA modification pathway in E. coli, these studies set a stage for work in higher organisms which in the long term will help to understand the specific roles of rRNA modification steps in biogenesis and their relevance to diseases.